In the past year, this project has consisted of testing multiple factors that could affect the function of chimeric antigen receptors (CARs). Chimeric antigen receptors consist of several components, the antigen-recognition moiety that is usually derived from a monoclonal antibody, a extracellular region that connects the antigen-recognition moiety to the transmembrane portion, costimulatory domains such as 4-1BB and CD28, and T cell activation domains such as CD3-zeta. We have constructed 10 new CARs over the past 6 months to test various components of CARs. T cells are transduced with the various CARs by using a gammaretroviral vector, and in vitro assays are carried out. The aim is to find CARs that impart T cells with the ability to kill cancer cells and proliferate without producing large amounts of potentially toxic inflamatory cytokines. We have found that changing the hinge region, costimulatory domains, or T cell activation domains all cause profound differences in CAR function. Following extensive in vitro testing, we will test promising CARs in a murine model within the next few months. This work has lead to a new fully-human anti-CD19 CAR for clinical testing that will start within the next 2-3 months years. Another aspect of this project is identification of new targets for CARs. We are assessing multiple targets that are expressed by leukemia and lymphoma as a main part of this project. Test of different CAR signaling chains and